EP2269594B1 - Verfahren zur herstellung eines thrombin-immobilisierten biologisch absorbierbaren folienpräparats - Google Patents

Verfahren zur herstellung eines thrombin-immobilisierten biologisch absorbierbaren folienpräparats Download PDF

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EP2269594B1
EP2269594B1 EP09733482.5A EP09733482A EP2269594B1 EP 2269594 B1 EP2269594 B1 EP 2269594B1 EP 09733482 A EP09733482 A EP 09733482A EP 2269594 B1 EP2269594 B1 EP 2269594B1
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Prior art keywords
thrombin
solution
sheet
bioabsorbable sheet
bioabsorbable
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French (fr)
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EP2269594A4 (de
EP2269594A1 (de
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Ryoichi Kawamura
Takayuki Imamura
Hitomi Owaki
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Chemo Sero Therapeutic Research Institute Kaketsuken
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Chemo Sero Therapeutic Research Institute Kaketsuken
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/42—Use of materials characterised by their function or physical properties
    • A61L15/44—Medicaments
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/42—Use of materials characterised by their function or physical properties
    • A61L15/64—Use of materials characterised by their function or physical properties specially adapted to be resorbable inside the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L31/148—Materials at least partially resorbable by the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L31/16—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/21—Acids
    • A61L2300/214—Amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/418—Agents promoting blood coagulation, blood-clotting agents, embolising agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/80—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special chemical form
    • A61L2300/802—Additives, excipients, e.g. cyclodextrins, fatty acids, surfactants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/04—Materials for stopping bleeding

Definitions

  • the present invention relates to a process for preparing a bioabsorbable sheet preparation holding thrombin. Specifically, the present invention relates to a process for preparing a bioabsorbable sheet preparation holding thrombin which comprises immersing a bioabsorbable support in a sheet form in a solution containing thrombin as an active ingredient, a polyhydric alcohol, a detergent, an amino acid and an oligosaccharide as an additive followed by drying and a bioabsorbable sheet preparation holding thrombin prepared by the process.
  • Thrombin is an enzyme that is involved in blood coagulation and is indispensable to maintenance and progress of life such as formation of hemostatic thrombus or treatment of injury.
  • Thrombin is normally present in blood in the form of inactive prothrombin but is activated by the coagulation system triggered by, for instance, platelets or damage in tissue cells to convert fibrinogen in a soluble form into insoluble fibrin useful for hemostasis and treatment of injury.
  • a hemostatic utilizing this principle has widely been applied in clinical scenes.
  • thrombin has been used as an active ingredient of a hemostatic, with the conventional hemostatic in the form of liquid, hemostasis by pressing or closing by pressing to a spot of gushing hemorrhage or oozing hemorrhage is not possible and therefore bleeding cannot be stopped.
  • a unique product of a sheet-type fibrin adhesive that has been commercially available is one where fibrinogen and bovine thrombin are held on a support consisting of equine collagen (product name: TachoComb/CSL Behling) (e.g. see Patent reference 8).
  • this adhesive still has room for improvement in handling and removal of risk factors in view of its thickness (about 3 mm) and inclusion of components derived from animals.
  • a stabilizing agent to thrombin comprising a salt of an organic carboxylic acid (e.g. see Patent reference 9) or glycerol (e.g. see Patent references 10, 11 and 12) for a liquid preparation, or by comprising a sugar, a basic amino acid and a liquid organic acid (e.g. see Patent reference 13), or gelatin, glycine and a sugar (e.g. see Patent reference 14), or an aliphatic polybasic carboxylic acid and albumin (e.g. see Patent reference 15), or human serum albumin and an amino acid (e.g. see Patent reference 16) for a dry preparation packed in a vial or in an aluminum pouch package.
  • a sheet-type preparation mere stabilization of thrombin cannot attain an object.
  • the thrombin preparation comprising a sugar, a basic amino acid and a liquid organic acid as described above is not suited for a sheet-type preparation since flexibility is lost when the thrombin preparation is held on a sheet.
  • a non-woven fabric is immersed in a thrombin solution and is simply subject to lyophilization, thrombin is not held on the non-woven fabric.
  • a technique is reported that a non-woven fabric made of chitin as a support is immersed alternately in three solutions, i.e.
  • EP1563856 discloses bioabsorbable synthetic nonwoven fabric comprising thrombin and hemostatic comprising said nonwoven fabric.
  • the nonwoven fabric can be prepared by dissolving thrombin in a saline or a buffer, adding optionally polyethylene glycol (detergent), arginine (amino acid), glycerol, mannitol (oligosaccharide), etc., immersing a nonwoven fabric into the solution, freezing at -80°C and lyophilizing.
  • an object of the present invention is to provide an expedient process for preparing a bioabsorbable sheet preparation holding thrombin and a bioabsorbable sheet preparation holding thrombin obtained by said process.
  • the present inventors have assiduously investigated, and as a result, have found that an excess production of lyophilized powder could be inhibited, and flexibility of a sheet preparation could be maintained, by comprising glycerol in a thrombin solution, and as a consequence, that the loss or destruction of thrombin constituent on a non-woven fabric due to fugacity of powder after drying could be avoided.
  • the present inventors have found that addition of Tween 80 enhanced permeation of thrombin into a non-woven fabric and that addition of trehalose and a small amount of histidine markedly increased stability of thrombin as compared to separate addition of trehalose or histidine, each alone, when a dry preparation is produced. As such, the present inventors have completed the present invention. Thus, the present invention is as defined by the claims.
  • a bioabsorbable sheet preparation holding thrombin and a process for preparing the same are provided.
  • the bioabsorbable sheet preparation holding thrombin of the present invention may be prepared, for instance, by immersing a bioabsorbable sheet consisting of polyglycolic acid in a thrombin solution comprising glycerol, Tween 80, trehalose and a small amount of histidine, followed by lyophilization.
  • glycerol By comprising glycerol, dropping off of thrombin constituent from a bioabsorbable sheet may be avoided and a bioabsorbable sheet preparation holding thrombin is endowed with flexibility.
  • thrombin permeation of thrombin into a bioabsorbable sheet may be enhanced to facilitate holding of thrombin.
  • trehalose and a small amount of histidine the activity of thrombin may sufficiently be maintained when a dry preparation is produced. Accordingly, the present invention may facilitate production of a bioabsorbable sheet preparation holding thrombin.
  • a bioabsorbable sheet preparation holding thrombin as prepared by the process of the present invention may possess stability of thrombin and flexibility necessary for a bioabsorbable sheet preparation.
  • thrombin may exert its enzymatic activity immediately after a sheet holding thrombin is in contact with liquid fibrinogen or blood.
  • thrombin may be eluted rapidly at the surface to be closed by adhesion or at active bleeding spot to exert its enzymatic activity with fibrinogen as a substrate to promptly provide fibrin conversion, providing tissue adhesion/closing effect and hemostatic effect.
  • the present invention is characterized by a process for preparing a bioabsorbable sheet preparation holding thrombin, which process utilizes a solution comprising a polyhydric alcohol, which is effective as a softening agent for the preparation, a detergent, which serves as enhancing permeation of thrombin into a bioabsorbable sheet, and a specific amino acid and an oligosaccharide, which maintains stability of thrombin, and a.bioabsorbable sheet preparation holding thrombin obtained by said process.
  • a process for preparing a bioabsorbable sheet preparation holding thrombin which process utilizes a solution comprising a polyhydric alcohol, which is effective as a softening agent for the preparation, a detergent, which serves as enhancing permeation of thrombin into a bioabsorbable sheet, and a specific amino acid and an oligosaccharide, which maintains stability of thrombin, and a.bioabsorbable sheet preparation holding thrombin obtained
  • a thrombin constituent as used herein may be either thrombin commercially available (e.g. Thrombin from human Plasma (Sigma-Aldrich, code No. T6884 etc.)), or thrombin from the living body as prepared from animal blood by a low-temperature ethanol fractionation or chromatography, or a recombinant thrombin obtained by a recombinant DNA technique.
  • a recombinant thrombin may be used so as to exclude contamination with unknown infectious factors or risk factors.
  • a thrombin gene may preferably be taken from an animal species to which hemostasis is to be applied.
  • thrombin In the living body, thrombin is synthesized as a precursor prothrombin in a vitamin K-dependent manner in hepatocytes and is subject to limited degradation by FXa-FVa on phospholipids of the cellular membrane (cleavage occurs at two sites, Arg320-ILe321 and Arg2.71-Thr272, of prothrombin) to generate thrombin.
  • FXa-FVa phospholipids of the cellular membrane
  • prothrombin is obtained by a recombinant DNA technique
  • expression may firstly be done for prothrombin which is, after purification, treated with ecarin to convert into thrombin.
  • a human prothrombin gene may be prepared starting from a whole RNA, mRNA or a genomic DNA in accordance with a general recombinant DNA technique as taught by Sambrook et al., Molecular Cloning, A Laboratory Manual Second Edition. Cold Spring Harbor Laboratory Press, N.Y., 1989 . Today, various kits are commercially available and may be used.
  • reagents such as TRIzol reagent (Invitrogen), ISOGEN (NIPPON GENE CO., LTD.), StrataPrep Total RNA Purification Kit (TOYOBO) for extraction of RNA; kits such as mRNA Purification Kit (Amersham Bioscience), Poly(A) Quick mRNA Isolation Kit (TOYOBO), mRNA Separator Kit (Clontech) for purification of mRNA; T-Primed First Strand Kit (Amersham Pharmacia), SuperScript plasmid system for cDNA synthesis and plasmid cloning (Invitrogen), cDNA Synthesis Kit (TAKARA SHUZO CO., LTD.), SMART PCR cDNA Synthesis & Library Construction. Kits (Clontech), Directionary cDNA Library Construction systems (Novagen Inc.), GeneAmp PCR Gold (Applied Biosystems) for conversion into cDNA may be used.
  • mRNA Purification Kit Amersham Bioscience
  • the thus obtained human thrombin gene may be inserted into a suitable expression vector and transformation of a host cell, e.g. an animal cell, with the resultant expression vector may be conducted.
  • An expression vector as used herein is not particularly limited but may be added with an expression regulator region such as a promoter suitable for an exogenous gene, termination codon, poly A addition signal sequence, kozack sequence, secretion signal, and the like.
  • a promoter contained in said expression vector may be any insofar as it leads to expression of an exogenous gene, such as an SV40 early promoter, an SV40 late promoter, a Cytomegalovirus promoter, a chicken ⁇ -action promoter, as selected based on an animal cell to be used as a host.
  • a chicken ⁇ -actin promoter-based expression plasmid pCAGG ( Japanese patent publication No. 3-168087 ) may be used.
  • a marker gene for selection or gene amplification may be one commonly known in the art such as a neo gene, a dihydrofolate reductase (dhfr) gene, a puromycin resistant enzyme gene, or a glutamine synthetase (GS) gene.
  • a host as used herein may be any culture cell from various mammals and includes Chinese hamster ovary cell (CHO cell), 293 cell derived from human, cells derived from chicken, and the like. Gene introduction into an animal cell may be performed, with no specific limitation, by e.g. a phosphate calcium method, a DEAE dextran method, a method using lipofectin liposome, a protoplast polyethylene glycol fusion, electroporation etc., which may suitably be selected depending on a host cell as used ( Molecular Cloning (3rd Ed.), Vol. 3, Cold Spring Harbor Laboratory Press (2001 )).
  • a medium to be used in culture includes an agar medium, a liquid medium, as classified from its form, or YMM-01, DMEM, RPMI, ⁇ MEM, etc. and may suitably be selected depending on a cell, the purpose of culture or a stage of culture.
  • a culture medium may be used in which sera, amino acids, vitamins, sugars, antibiotics, pH adjusting buffers, and the like are added. pH of media may be adjusted in a range of 6-8 and a culture temperature may be in a range of 30°C to 39°C. An amount of a medium, any additive and a culture period may suitably be adjusted depending on a culture scale.
  • Cells producing a human prothrombin may be obtained by detecting a human prothrombin present in a culture solution of the cloned drug-resistant cells by a detection method utilizing a specific reaction with an anti-thrombin antibody such as dot blot, Western blot, sandwich ELISA, etc.
  • the thus obtained cells producing a human prothrombin may be adapted to a serum-free culture medium and then subject to culture in a large amount at a level of industrial production. Culture in a large amount may be done by e.g. fed batch culture, batch culture, etc. with no specific limitation.
  • a purification method as generally used in protein chemistry may be used such as e.g. a salting out, a ultrafiltration, an isoelectric precipitation, an electrophoresis, an ion exchange chromatography, a gel filtration chromatography, an affinity chromatography, an hydrophobic chromatography, a hydroxyapatite chromatography, etc.
  • a combination of the above methods may sometimes be employed due to presence of a variety of cellular debris.
  • a recombinant ecarin may preferably be used for conversion of human prothrombin into human thrombin.
  • a recombinant ecarin may be prepared as described in the patent publication ( WO2003/004641 ). Briefly, a snake venom ecarin cDNA is prepared as taught by Nishida et al. ( S. Nishida et al., Biochemistry, 34, 1771-1778, 1995 ) and incorporated into a chicken ⁇ -actin promoter-based expression plasmid pCAGG as used herein. The obtained expression vector is introduced into animal cells, e.g. CHO cells, to provide cells producing a recombinant ecarin.
  • a recombinant ecarin may be purified by a cation exchange chromatography and a gel filtration. The thus purified recombinant ecarin may be acted to a human prothrombin for conversion into human thrombin.
  • the reaction condition may be the same as that of a normal enzymatic reaction. For instance, ecarin at a final concentration of 2-8 Units/mL may be added to a human prothrombin at 1000 ⁇ g/mL for reaction at 36-38°C for 1-4 hours to complete conversion of a human prothrombin into a human thrombin.
  • human thrombin is purified from the solution after treatment with ecarin, it may be done by subjecting the solution to the method for purifying a protein as described above.
  • a method for purification using a combination of a benzamidine chromatography and a cation exchange chromatography may be used.
  • human thrombin may be adsorbed to a column equilibrated with a buffer containing 0.3-0.7 M NaCl, pH 7-9, and after washing with the buffer, may be eluted with the buffer supplemented with 0.1 M benzamidine hydrochloride.
  • the above eluate of human thrombin may be subject to a cation exchange chromatography.
  • a cation exchanger such as a sulfopropyl type or a carboxymethyl type has been developed and may be selected as appropriate for use.
  • SP Toyopearl 550C (Tosoh Corporation) is used in the present invention.
  • Human thrombin may be adsorbed to a column equilibrated with 10 mM citrate buffer containing 0.2 M NaCl, pH 6-7, and after washing with the buffer, may be eluted with the buffer containing 0.6 M NaCl. By these procedures, a human thrombin of high purity may be obtained.
  • Unit of activity of the thus obtained human thrombin may be indicated by clotting activity and the activity to cleave a synthetic substrate (S-2238).
  • a value for the clotting activity represents a time required for clotting by fibrinogen relative to a calibration curve of standard, including thrombin of the Japanese Pharmacopoeia standard and WHO international standard (NIBSC).
  • the activity to cleave S-2238 which is based on a reaction between thrombin and its specific substrate, may be measured by determining an amount of p-nitroaniline released upon cleavage of the synthetic substrate S-2238 by thrombin through a change in absorbance at OD405/650.
  • a bioabsorbable sheet may be in any form insofar as flexibility and elasticity are ensured to some extent such as a textile, woven fabric or non-woven fabric prepared by processing a bioabsorbable synthetic fiber into sheet.
  • a bioabsorbable synthetic fiber may be selected from the group consisting of polyglycolic acid, polylactic acid, carboxymethyl cellulose, chitin, chitosan, alginic acid, collagen, gelatin and hydrogel.
  • polyglycolic acid non-woven fabric may be used which is prepared by knitting or weaving polyglycolic acid as a substrate and needle-punching the knit or fabric to prepare a non-woven fabric.
  • thrombin solution a solution used for holding thrombin on a bioabsorbable sheet
  • thrombin solution may be added not only thrombin as an active ingredient of a hemostatic but also a stabilizing agent for maintaining the activity of thrombin per se, a softening agent for avoiding the loss or fugacity of thrombin constituent held on a bioabsorbable sheet, and a permeating agent for enhancing permeation of thrombin into a bioabsorbable sheet.
  • Thrombin may be contained in a thrombin solution at such a concentration that the hemostatic activity is exerted, e.g. at a final concentration of 100 to 5,000 Units/mL, preferably at 1,000 to 2,000 Units/mL.
  • the loss or decrease in the activity of thrombin is due to aggregation of thrombin while storage.
  • a stabilizing agent of thrombin a substance capable of inhibiting aggregate formation of thrombin may be used.
  • Such a substance is a specific amino acid, namely histidine.
  • a final concentration of the amino acid may be in a range of 2.4 to 180 mM.
  • An oligosaccharide may be used that effectively maintains the activity of thrombin while storage.
  • Such an oligosaccharide is trehalose.
  • trehalose if supplied to the organ surface at surgical operation, may inhibit denaturation of the tissue and to prevent dry adhesion (http://www.vm.a.u-tokyo.ac.jp/vmc/achievement/ treharose.html, or http://www.next21.info/press/images/ 20050624Treha.pdf). Therefore, trehalose is expected to be effective not only as a stabilizing agent but also for decreasing adhesion of the surface to be treated for closure with other organs. Thus, trehalose may preferably be used. A final concentration of an oligosaccharide may be in a range of 5 to 40 mg/mL.
  • a thrombin solution of the present invention may contain both an amino acid and an oligosaccharide and a ratio of these two may suitably be adjusted within the ranges as described above.
  • an amino acid at 22.5 to 180 mM and an oligosaccharide at 30 to 40 mg/mL may be used.
  • the most preferable embodiment is histidine at 180 mM and trehalose at 40 mg/mL.
  • a softening agent and a permeating agent may be used that may not affect the activity of thrombin and that may maintain flexibility of a bioabsorbable sheet.
  • a softening agent is glycerol. It has efficaciously been used as a stabilizing agent in various medicaments and vaccines.
  • a softening agent may be used at a final concentration in a range of 1 to 2%.
  • a detergent may be used such as Tween 80, poly(oxyethylene) lauryl ether (Brij 35), Pluronic F-68, Sucrose monolaurate, Sodium Cholate, Tween 20, Triton X-100, Nonidet P40, sulfuric-3-[(3-cholamidpropyl)dimetylammonio]-1-propane (CHAPS), n-Octylglucoside, n-Dodecylmaltoside and Digitonin, preferably Tween 80.
  • a permeating agent may be used at a concentration in a range of 0.01 to 1%, preferably 0.01 to 0.1%.
  • a softening agent and a permeating agent of the present invention may be used not only in a bioabsorbable sheet preparation holding thrombin but also in a bioabsorbable sheet preparation holding other hemostatic proteins, e.g. fibrinogen.
  • a buffer used for preparing a thrombin solution may include a phosphate buffer, Tris buffer, a citrate buffer, and the like, at a pH range of 6.0 to 8.0.
  • Other additives such as a salt, e.g. calcium chloride, sodium chloride, or excipients such as sucrose and mannitol in an appropriate amount may be added as appropriate.
  • a bioabsorbable sheet preparation holding thrombin of the present invention may be obtained, for instance, by adding dropwise a thrombin solution to a bioabsorbable sheet consisting of polyglycolic acid put in an appropriate container or immersing the bioabsorbable sheet in a thrombin solution in a container and, after lyophilization at -80°C, drying the sheet.
  • a bioabsorbable sheet filled with a thrombin solution may be subject to natural drying.
  • permeability of thrombin into the bioabsorbable sheet may be enhanced.
  • thrombin may stably be held on the fibers of the bioabsorbable sheet wrapped in a stabilizing agent and a softening agent.
  • the obtained bioabsorbable sheet preparation holding thrombin may be stored at room temperature (22 to 25°C) with tight sealing and packaging in a two-layered (polyethylene-aluminum) film pack together with a drying agent.
  • Estimation of the thus obtained bioabsorbable sheet preparation holding thrombin may be carried out by testing a holding rate of thrombin held on a bioabsorbable sheet, flexibility of a bioabsorbable sheet preparation holding thrombin after molding, stability of thrombin, and the like.
  • a holding rate of thrombin on a bioabsorbable sheet may be calculated by comparing the activity of thrombin eluted from the sheet after immersing the sheet in a saline or a suitable buffer and shaking with the activity of thrombin held on the sheet.
  • the clotting activity and the activity to cleave a synthetic substrate as described above may be used.
  • a bioabsorbable sheet preparation holding thrombin with a holding rate of 80% or more may be obtained.
  • Flexibility of a bioabsorbable sheet preparation holding thrombin of the present invention may be estimated as described below. Namely, a bioabsorbable sheet holding thrombin is cut into an appropriate piece and one end of the piece is fixed on a pedestal. A fibrinogen solution prepared at about 55 mg/mL is then added dropwise to the portion horizontally projecting from the pedestal. The fibrinogen is immediately converted into fibrin by the action of thrombin held on the bioabsorbable sheet to let the sheet be hanged downward by the weight of fibrin. Flexibility may be estimated by measuring how much the tip of the projecting portion is hanged down from the horizontal level line.
  • Stability of a bioabsorbable sheet preparation holding thrombin of the present invention may be estimated by evaluating the remnant enzymatic activity of thrombin and an extent of aggregation or degradation of a thrombin molecule after storage for a long period of time.
  • the remnant enzymatic activity of thrombin may be tested by measuring the enzymatic activity in the same manner as in the test of a holding rate of thrombin as described above.
  • An extent of aggregation or degradation of a thrombin molecule may be tested by subjecting a portion of thrombin eluted from the sheet to SDS-PAGE or size exclusion high performance liquid chromatography.
  • a bioabsorbable sheet preparation holding thrombin of the present invention may be used as an adhesive by applying the preparation to a spot of injury or hemostasis. Depending on severity of bleeding, the bioabsorbable sheet preparation holding thrombin may be applied either alone or together with fibrinogen, a constituent of a fibrin adhesive preparation.
  • a bioabsorbable sheet preparation holding thrombin may be applied alone to the affected surface followed by pressing for 3 to 5 minutes to accomplish hemostasis.
  • fibrinogen a constituent of a fibrin adhesive preparation, is applied together, the closing effect or the hemostatic effect may be enhanced.
  • air leakage site may effectively be closed at a peeled surface of the lung pleura where expansion and contraction pressures of the lung are generated, a cut surface of the parenchyma of the lung lobe, or a cut edge of the bronchia.
  • hemostatic treatment becomes extremely difficult due to indefiniteness of bleeding points but the present invention makes it possible to close a fixed area at a time with a single treatment.
  • the present invention may exert the utmost effect, while maintaining a sufficient amount of fibrinogen at te affected site, to allow for formation of fibrin promptly and rigidly to thereby accomplish hemostasis.
  • a bioabsorbable sheet preparation holding thrombin of the present invention is highly flexible and hence may follow the unevenness of the tissue so as to be tightly adhered to and that thrombin is promptly eluted to convert fibrinogen into fibrin.
  • a thrombin solution (pH 6.0) containing glycerol at a final concentration of 1% or 1.5% or 2%, 1875 Units/mL thrombin, 10 mg/mL human serum albumin, 40 mM calcium chloride, 5 mg/mL mannitol and 0.1% Tween 80 was poured into a container, laid with a bioabsorbable sheet (a polyglycolic acid bioabsorbable synthetic non-woven fabric, Product name: Neoveil (Gunze Limited)), at 1 mm of thickness and, after lyophilization at -80°C for 2 hours, dried in vacuum to prepare a bioabsorbable sheet preparation holding thrombin. This preparation was compared with a bioabsorbable sheet preparation holding thrombin prepared in the same manner as described above but with a thrombin solution not containing glycerol.
  • a bioabsorbable sheet a polyglycolic acid bioabsorbable synthetic non-woven fabric, Product name: Neove
  • a drying agent with tight sealing and packaging and stored at room temperature (22 to 25°C).
  • the sample packages were opened, the bioabsorbable sheet preparations holding thrombin were peeled off from the containers and cut into pieces of 2 cm ⁇ 2 cm (4 cm 2 ) which were immersed in a saline (1.0 mL) with thorough mixture to let thrombin be eluted.
  • a clotting time was defined as a time required, after mixing a fibrinogen solution and a thrombin solution, for attaining 0.3 of absorbance (450 nm) of the resulting solution containing fibrin.
  • a standard solution four kinds of units prepared with thrombin standard of applicant's own was mixed with a fibrinogen solution at a fixed concentration and a clotting time was measured.
  • a standard line showing a relationship between unit of activity of thrombin and a clotting time was prepared where the axis of abscissas indicates the unit of activity and the axis of ordinates indicates the clotting time.
  • thrombin eluate A clotting time was measured in like manner for the above thrombin eluate and, using the standard line, unit of activity of thrombin was obtained and unit of remnant activity of thrombin per area of the sheet (Unit/cm 2 ) was calculated.
  • Fig. 2 shows the remnant activity of thrombin in samples with and without addition of glycerol where the axis of abscissas indicates a period of time for storage and the axis of ordinates indicates unit of clotting activity (Unit/cm 2 ) of the thrombin eluate after storage as compared to that before storage.
  • bioabsorbable sheet preparations holding thrombin were prepared with tight sealing and packaging and stored at 37°C as described in Examples 1 and 2.
  • bioabsorbable sheet preparations holding thrombin were prepared with tight sealing and packaging and stored at 65°C as described in Examples 1 and 2.
  • oligosaccharide in Table 1 sucrose, trehalose, sorbitol, erythritol, lactose, and as a control, mannitol, which is a constituent previously reported, were used. Eight days after storage, the sample packages were opened, the thrombin eluate was obtained and the remnant activity of thrombin as maintained relative to the activity before storage was measured and indicated in graph as shown in Fig. 4 .
  • sucrose, trehalose and sorbitol exhibited the equivalent stabilizing effect on thrombin to mannitol.
  • Table 1 Thrombin solution Thrombin 1,000 Units/mL Oligosaccharide 10 mg/mL Glycerol 1% Human serum albumin 10 mg/mL Tween 80 0.1% Citrate buffer 50 mM, pH 6.0
  • bioabsorbable sheet preparations holding thrombin were prepared with tight sealing and packaging and stored at 65°C as described in Examples 1 and 2. Three days after storage, the sample packages were opened, the thrombin eluate was obtained and the remnant activity of thrombin was measured.
  • mannitol and trehalose were used for the oligosaccharide in Table 2. As shown in the graph of Fig. 5 , it was proved that, at a high concentration of 8 mg/mL or more, trehalose was more excellent in the effect of maintaining the activity than mannitol.
  • bioabsorbable sheet preparations holding thrombin were prepared with tight sealing and packaging and stored at 65°C as described in Examples 1 and 2.
  • amino acid in Table 3 glycine as a simple amino acid, glutamic acid and aspartic acid as an acidic amino acid, arginine, histidine and lysine as a basic amino acid were used.
  • a thrombin solution without addition of an amino acid and a thrombin solution containing human serum albumin in place of an amino acid were used.
  • the sample packages were opened, the thrombin eluate was obtained and the activity of thrombin was measured to obtain the remnant activity relative to the activity of the eluate before storage.
  • Control 1 without addition of amino acid
  • Control 2 Thrombin 1,000 Units/mL 1,000 Units/mL 1,000 Units/mL
  • bioabsorbable sheet preparations holding thrombin were prepared with tight sealing and packaging and stored at 65°C as described in Examples 1 and 2.
  • a citrate buffer was not added for the thrombin solutions with addition of histidine. Eight days after storage, the sample packages were opened, the thrombin eluate was obtained and the activity of thrombin was measured to obtain the remnant activity after storage.
  • the remnant activity of thrombin was as low as 0.8 to 2.4% when trehalose at 5 to 40 mg/mL or histidine at 2.4 to 90 mM was added each alone but the remnant activity increased when trehalose and histidine were added simultaneously as compared to the addition of trehalose or histidine each alone, depending on a concentration the respective constituents.
  • the remnant activity increased to 5.6% to 23.1% by adding histidine at 90 to 180 mM.
  • the remnant activity increased to 6.6% to 36.4% by adding histidine at 22.5 to 180 mM.
  • the remnant activity increased to 21.4% to 62.0% by adding histidine at 2.4 to 180 mM.
  • the effect of addition of histidine was remarkable when trehalose was at a higher concentration.
  • trehalose was at 30 to 40 mg/mL, a synergetic effect could be seen even when histidine was added at a lower concentration of 2.4 mM.
  • a synergetic effect was prominent when histidine was added at 22.5 to 180 mM where the remnant activity of thrombin increased to 35% or more.
  • the bioabsorbable sheet described in Example 1 was immersed in a thrombin solution with the composition as shown in Table 5 for 15 seconds and increase in a weight of the sheet after immersion was measured.
  • Table 5 For the detergent in Table 5, Tween 80, poly(oxyethylene) lauryl ether (Brij 35), Pluronic F-68, Sucrose monolaurate, Sodium Cholate, Tween 20, Triton X-100, Nonidet P40, sulfuric-3-[(3-cholamidpropyl)dimetylammonio]-1-propane (CHAPS), n-Octylglucoside, n-Dodecylmaltoside and Digitonin were used.
  • Tween 80 poly(oxyethylene) lauryl ether (Brij 35), Pluronic F-68, Sucrose monolaurate, Sodium Cholate, Tween 20, Triton X-100, Nonidet P40, sulfuric-3-[(3-cholamidpropyl)d
  • Thrombin filler solution Control Thrombin 1,000 Units/mL 1,000 Units/mL Amino acid (histidine) 180 mM 180 mM Oligosaccharide (Trehalose) 30 mg/mL 30 mg/mL Glycerol 1% 1% Calcium chloride 20 mM 20 mM Detergent 0.1% No addition
  • the bioabsorbable sheet described in Example 1 was immersed in a thrombin solution with the composition as shown in Table 5 containing as a detergent Tween 80 at various concentrations for 15 seconds and increase in a weight of the sheet after immersion was measured. Final concentrations of 0.0001, 0.001, 0.01, 0.1 and 1% of Tween 80 were used. As a control, the thrombin solution as shown in Table 5 with no addition of a detergent was used.
  • thrombin solution with the composition as shown in Table 6, flexibility of the bioabsorbable sheet preparation holding thrombin prepared as described in Example 1 was estimated.
  • a portion (2 cm ⁇ 1 cm) of the sheet (2 cm ⁇ 3 cm) of the present invention was fixed on a pedestal and a fibrinogen solution at 55 mg/mL was added dropwise to the remaining portion (2 cm ⁇ 2 cm) of the sheet horizontally projecting from the pedestal. A distance and an angle of the tip of the projecting portion as being hanged down from the horizontal level line were measured to estimate flexibility.
  • a collagen sheet holding the constituent of a fibrin adhesive product name: TachoComb/CSL Behling
  • control collagen sheet preparation Since the control collagen sheet preparation has fibrinogen being previously held thereon by drying, a saline was used for the solution to be added dropwise and a test was performed under condition where the adhesive surface is set upside. The measurement was made after being left to stand for 5 minutes after dropwise addition of the solution and a mean value and standard deviation of three measurements were indicated in graph as shown in Fig. 11 .
  • control collagen sheet preparation showed a distance of hanging down of less than 5 mm at the time when 0.1 mL of the saline was added dropwise whereas the sheet of the present invention was so flexible that it has already been hanging down for its own weight before dropwise addition of the fibrinogen solution and showed a distance of hanging down of about 20 mm after dropwise addition of 0.1 mL of the fibrinogen solution.
  • the sheet hanged down too extensively to measure the distance When 0.3 mL of the fibrinogen solution was added dropwise, the sheet hanged down too extensively to measure the distance.
  • Thrombin filler solution Thrombin 1,875 Units/mL Amino acid (histidine) 180 mM Oligosaccharide (trehalose) 30 mg/mL Glycerol 1% Calcium chloride 20 mM Detergent (Tween 80) 0.1% Citrate buffer 50 mM, pH 6.0
  • a bioabsorbable sheet preparation holding thrombin and a process for preparing the same are provided.
  • the bioabsorbable sheet preparation holding thrombin of the present invention may be prepared, for instance, by immersing a bioabsorbable sheet consisting of polyglycolic acid in a thrombin solution comprising glycerol, Tween 80, trehalose and a small amount of histidine, followed by lyophilization.
  • glycerol By comprising glycerol, dropping off of thrombin constituent from a bioabsorbable sheet may be avoided and a bioabsorbable sheet preparation holding thrombin is endowed with flexibility.
  • thrombin permeation of thrombin into a bioabsorbable sheet may be enhanced to facilitate holding of thrombin.
  • trehalose and a small amount of histidine the activity of thrombin may sufficiently be maintained when a dry preparation is produced. Accordingly, the present invention may facilitate production of a bioabsorbable sheet preparation holding thrombin.
  • a bioabsorbable sheet preparation holding thrombin as prepared by the process of the present invention may possess stability of thrombin and flexibility necessary for a bioabsorbable sheet preparation.
  • thrombin may exert its enzymatic activity immediately after a sheet holding thrombin is in contact with liquid fibrinogen or blood.
  • thrombin may be eluted rapidly at the surface to be closed by adhesion or at active bleeding spot to exert its enzymatic activity with fibrinogen as a substrate to promptly provide fibrin conversion, providing tissue adhesion/closing effect and hemostatic effect.

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Claims (14)

  1. Verfahren zur Herstellung eines bioresorbierbaren Folienpräparats, das Thrombin enthält, das die folgenden Schritte (1) bis (3) umfasst:
    (1) Schritt des Herstellens einer Lösung, umfassend ein Detergens, Trehalose, Histidin, Glycerin und Thrombin
    (2) Schritt des tröpfchenweise Hinzufügens der Lösung aus (1) zu einer bioresorbierbaren Folie oder des Eintauchens einer bioresorbierbaren Folie in die Lösung aus (1); und
    (3) Schritt des Trocknens der bioresorbierbaren Folie aus (2).
  2. Verfahren nach Anspruch 1, wobei das Detergens Polyoxyethylen(80)-sorbitanmonooleat (Tween 80) ist.
  3. Verfahren nach Anspruch 1 oder 2, wobei Thrombin in einer Konzentration von 1000 bis 2000 Einheiten/ml vorliegt.
  4. Verfahren nach Anspruch 2, wobei Polyoxyethylen(80)-sorbitanmonooleat (Tween 80) mit 0,01 bis 1% vorliegt.
  5. Verfahren nach Anspruch 1 oder 2, wobei Histidin im Bereich von 2,4 bis 180 mM vorliegt.
  6. Verfahren nach Anspruch 1 oder 2, wobei Trehalose im Bereich von 5 bis 40 mg/ml vorliegt.
  7. Verfahren nach Anspruch 1 oder 2, wobei Glycerin in einer Konzentration von 1 bis 2% vorliegt.
  8. Bioresorbierbares Folienpräparat, das Thrombin enthält und das ein Detergens, Trehalose, Histidin, Thrombin und einen Polyalkohol, bevorzugt Glycerin, umfasst.
  9. Präprarat nach Anspruch 8, wobei das Detergens Polyoxyethylen(80)-sorbitanmonooleat (Tween 80) ist.
  10. Präparat nach Anspruch 8 oder 9, wobei Thrombin in einer Konzentration von 1000 bis 2000 Einheiten/ml vorliegt.
  11. Präparat nach Anspruch 9, wobei Polyoxyethylen(80)-sorbitanmonooleat (Tween 80) mit 0,01 bis 1% vorliegt.
  12. Präparat nach Anspruch 8 oder 9, wobei Histidin im Bereich von 2,4 bis 180 mM vorliegt.
  13. Präprarat nach Anspruch 8 oder 9, wobei Trehalose im Bereich von 5 bis 40 mg/ml vorliegt.
  14. Präparat nach Anspruch 8 oder 9, wobei Glycerin in einer Konzentration von 1 bis 2% vorliegt.
EP09733482.5A 2008-04-16 2009-04-15 Verfahren zur herstellung eines thrombin-immobilisierten biologisch absorbierbaren folienpräparats Not-in-force EP2269594B1 (de)

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CN102105141B (zh) 2015-06-24
CA2721335A1 (en) 2009-10-22
US20110038847A1 (en) 2011-02-17
US9149557B2 (en) 2015-10-06
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